Lung cancer is the most common human neoplasm in the U.S. and, increasingly, in much of the world. While smoking is known to be the major etiological factor, the causative cellular and molecular mechanisms are complex and not well understood. Currently, we are focusing on three aspects of causation, behavior and possible new treatment responsiveness of adenocarcinoma, the most common form of lung cancer: (1) the role of the mutant K-ras oncogene, especially involvement of reactive oxygen species; (2) contributions of a signaling pathway triggered by the ErbB3 receptor and effects of siRNA-based therapy; and (3) ribosomal RNA as a potential therapeutic target. (1)Mutant K-ras and lung cancer. The oncogene K-ras is often mutated in adenocarcinoma of the lung (as well as other common carcinomas), but the wild-type form is tumor suppressive. An important question, then, is: why is mutant K-ras actively oncogenic? Answers to this question could aid in prevention of up to 50 percent of human lung adenocarcinomas, and an even higher percentage of cancers of the colon and pancreas. We have found that transfection of mutant K-ras in lung epithelial cells causes increases in reactive oxygen species and DNA damage. In a panel of cell lines derived from clinical human lung adenocarcinomas, DNA damage was increased relative to a nontransformed line, as measured by the comet assay for DNA strand breaks. The degree of this damage was significantly correlated with intracellular levels of superoxide, a reactive form of oxygen. Superoxide was measured by the nitroblue tetrazolium reduction assay. Experimental downregulation of superoxide reduced DNA damage. An enzyme, OGG1, which repairs specifically oxidative DNA damage, was negatively correlated with DNA damage. These results showed that oxidative DNA damage by superoxide is a consistent, but variable, property of human lung adenocarcinoma cells. Furthermore, for a subset of the cell lines, both DNA damage and superoxide were positively correlated with amounts of K-ras protein. Thus, the hypothesis that mutant K-ras contributes to lung malignancy through reactive oxygen species is supported. In our most recent findings, an alternative form of the mutant K-ras protein, termed K-ras4A, was found to be expressed highly in some of the lines, and to correlate strongly with superoxide production. This isoform may have a special, heretofore unsuspected, role. Overall, these results encourage use of antioxidants for prevention/intervention of lung cancer and possibly as supplementary therapy for established cancers. (2)The oncogene ErbB3 in lung cancer. The majority of human and mouse lung adenocarcinoma cell lines, but not nontransformed cells, express the ErbB3 receptor, which belongs to the epidermal growth factor receptor family. We have demonstrated that ErbB3 in the lung cancer cells signals through phosphatidylinositol 3-kinase, AKT, GSK3beta, and cyclin D1 to stimulate the cell cycle and also cell invasiveness and migration. These behaviors in cell culture can be blocked with siRNA to ErbB3 or to the several Akt isoforms. Thus, siRNA treatment may be an approach to therapy. To confirm this, it is necessary to test the siRNA on tumors transplanted in vivo into mice. ErbB3 or AKT2 siRNA markedly suppressed the growth of human lung adenocarcinoma xenografts, 60% to 80%, in four separate trials, with residual effects several weeks after the end of treatment. Notably, the siRNAs were administered as saline solutions. Previously it had been thought that siRNAs would be too unstable by this route to be effective. Elimination of the need for complex carriers is very hopeful, since these carriers can introduce many complications clinically. After some additional confirmatory work, we expect to introduce this siRNA approach into the pipeline for possible clinical application. (3) Ribosomal RNA as a therapeutic target in lung cancer. Ribosomal RNA (rRNA) is a limiting component of ribosomes and is highly regulated in its expression from its multiple gene copies. Ribosomes are necessary for cancer growth, and over-production of ribosomes may even drive cancer development. Using human lung adenocarcinoma cells, we have discovered that a noncoding (nc) RNA is transcribed from the rRNA gene complex, starting in the intergenic spacer region. This ncRNA had previously been described only in mouse fibroblasts. Thus we are the first to report it in human cells, in epithelial cells, and in cancer cells. Furthermore, in a panel of thirteen human lung epithelial cancer cell lines, the ncRNA amount (as determined by real-time PCR), correlated negatively with amount of total rRNA present in eleven of the lines. Thus it appears that the ncRNA may commonly have some sort of negative regulatory role controlling production of the rRNA and hence ribosomes. Furthermore, a single nucleotide polymorphism was present in the 5' leader region of the rRNA, with high frequency in several cell lines. This 5' leader region is key in initiating the processing of pre(45S)rRNA in the nucleus. Strikingly, the frequency of this gene polymorphism correlated negatively with amounts of ncRNA and positively with total rRNA. Although many mechanistic questions still need to be answered, it is possible that this polymorphism affects human risk of lung cancer. In another component of this project, we have studied the rRNA and ncRNA in mouse lung epithelial cells, both nontransformed and cancerous. To test whether the ncRNA, as a possible regulator of rRNA, could be a potential therapeutic target, we utilized an RNA-inhibitory oligonucleotide gapmer, a hybrid of DNA and locked nucleic acid (LNA) analogue with enhanced affinity to target sequences and capability for nuclear entry. This gapmer was targeted to the ncRNA. It had the exciting effect of reducing rRNA and causing cell death, and this effect was, importantly, much more pronounced in the malignant cells, compared with sister cell lines of nontransformed cells. This selectivity encourages thoughts of therapeutic use. We continue to study the mechanism of action of the ncRNA, the molecular sequence of greatest vulnerability for therapeutic targeting, and the cellular pathways leading to cell death. Once these details are understood, we can test the gapmer reagents in vivo. Also, we will be extending these efforts to human lung cancer cells.